In Vitro and In Vivo Effect of Xylopic Acid on Cytochrome P450 Enzymes

Introduction Xylopic acid (XA), the major constituent of the fruit of Xylopia aethiopica, has shown several pharmacological properties. Traditionally, the plant is used to treat several diseases and is being used in the preparation of several local foods despite the lack of information about its safety, food-drug interaction, and other pharmacokinetic properties. This study, therefore, investigated the effect of XA on rat liver cytochrome P450 (CYP) enzymes in vivo and in vitro. Methods Inhibition or induction of some isoforms of CYP450 enzymes: CYP 1A1/1A2, 1A2, 2B1/2B2, 3A4, 2D6, and 2C9 were investigated using microsomal fractions of the liver obtained from rats pretreated with a low dose of xylopic acid (LDT) 30 mg/kg, high dose of xylopic acid (HDT) 100 mg/kg, phenobarbitone (PC) 80 mg/kg, and ketoconazole (NC) 100 mg/kg, and a no-treatment group received distilled water, with (n = 5) animals in each group. The in vitro inhibition of CYP 3A4 was assessed by treating rat liver microsomes with XA. Results Xylopic acid induced CYP 1A1/1A2, 1A2, 2D6, and 2C9, inhibited CYP 3A4, and had no effect on 2B1/2B2. Conclusion The findings would help mitigate toxicity and therapeutic failure especially in cases of coadministration of medications with food containing XA, with metabolism altered by the latter.


Introduction
e use of medicinal natural products is traced back thousands of years due to the belief that herbal drugs have fewer side effects, enhance the effects of conventional agents, and can be an alternative for the management of several disorders [1,2]. An example of one of such herbs is the Xylopia aethiopica. Studies have shown that the plant has anti-inflammatory, antimicrobial, analgesic, antiparasitic, and antioxidant properties [3][4][5]. In Ghana, the fruit is used by traditionalists to manage a wide range of disorders as it is believed to have curative effects on dyspepsia, cough, pain, and parasitic infections. It is equally used as a spice in the preparation of dishes such as porridge (Hausa Koko), sobolo, other African cuisines, and soups [5,6]. Some food industries use it as a flavoring agent, and in some cosmetic industries, it is used as an agent for fragrance [7]. e plant is widely distributed in the West African rainforest from Senegal to Sudan in Eastern Africa and down to Angola in Southern Africa [8,9] where it is mostly used for local cooking, especially in the preparation of what is referred to as the African pepper soup [10]. e plant grows prominently in several parts of Africa including Ghana, Nigeria, Benin, Kenya, Ivory Coast, and several other places [11]. Several studies have been performed on the fruit, and these studies reveal the plant contains about 98 compounds [12,13]. Constituents of the fruit include essential oils, volatile oils, resin, arocene, rutheroside fat, bitter principles, alkaloids, glycosides, saponins, tannins, mucilage, xylopic acid, and kaurenoic acid [14]. e compounds obtained from the fruit that are considered most relevant are xylopic acid and kaurenoic acid. Xylopic acid, a kaurene diterpene, occurs as the major constituent in the fruits of Xylopia aethiopica [15].
Research on xylopic acid has shown that it exhibits diuretic and vasorelaxant properties in rodents [16]. Xylopic acid caused a substantial reduction of lung inflammation induced by carrageenan in mice, which supports the traditional use of the plant extracts of Xylopia aethiopica as therapeutic agents in conditions associated with acute inflammation and some respiratory disorders [17]. Xylopic acid possesses curative and prophylactic properties on P. berghei-induced malaria in ICR mice as well as antipyretic properties [18]. Xylopic acid and the fruit extract of Xylopia aethiopica were found to have significant central nervous system depressant effects in mice [3,19]. e chemical structure of xylopic acid is shown in Figure 1.
Generally, people use traditional medicine with the notion that it has little or no side effects. Hence, such herbs do not pass through rigorous tests before approval is granted compared to conventional drugs [20,21]. Due to insufficient scrutiny, most herbal medicines may result in CYP-mediated herb-drug interactions when coadministered with other orthodox medications, and this may go unnoticed [22]. Interactions of traditional medicines with human CYPs have been associated with alterations in the pharmacokinetics of several drugs [23]. ey interact by either inhibiting or inducing CYPs which may result in harmful side effects. Drug-drug and herb-drug interactions is an important phenomenon that should be investigated before new agents are introduced on the market. Hence, it is useful to conduct series of investigations on the effect of traditional medicines on liver CYP450 drug-metabolizing enzymes [20].
is study sought to evaluate the effect of xylopic acid on carefully selected CYPs that form the major drug-metabolizing enzymes in the human system.
Male rats weighing 250-300 g (n � 5) were randomly assigned to one of the four experimental groups, being lowdose xylopic acid treatment group (LDT) 30 mg/kg, highdose xylopic acid treatment group (HDT) 100 mg/kg, phenobarbitone (PC) 80 mg/kg, a no-treatment group that received distilled water, and finally for CYP 3A4, a control group treated with ketoconazole (KC) 100 mg/kg with (n � 5) animals in each group. e choice of dosage for XA was informed by previous studies on the interaction of XA on hepatic enzymes using pentobarbitone-induced sleep test in mice [24]. e treatment for each group was administered orally for 7 days. All the animals were anesthetized by CO 2 inhalation to minimize suffering, then sacrificed by abdominal venesection method after which their liver was harvested, snap-frozen in liquid nitrogen, and stored at −80°C until further analysis was carried out.

Preparation of Microsomal, Cytosolic
Fractions. Liver samples of 7.5 g were obtained from each animal and kept in the −80°C freezer until use. On the day of the experiment, each liver sample was then homogenized in two volumes of 20 mM potassium phosphate buffer (pH 7.4) using a mortar and a pestle. e homogenate was centrifuged at 2,500 rpm OAC COOH Figure 1: Chemical structure of xylopic acid [5].
for 50 minutes at 4°C and further centrifuged at 40,000 rpm for 60 minutes at 4°C. e resultant supernatant (cytosol) was then separated from the pellet (microsomes). e microsomes obtained were further homogenized in potassium phosphate buffer 20 mM (pH 7.4) and stored at −80°C [25,26].

Protein Determination and Standardization.
e protocol for protein determination was adapted from the protocol described by Harwood et al. [25,26]. Fourfold serial dilutions were performed on the microsomal solutions using potassium phosphate buffer. en, a twofold serial dilution for seven concentrations was prepared for a protein standard, bovine serum albumin (BSA). A volume of 10 μL of BSA and 200 μL of Bio-Rad reagent which was then added to each microsomal dilution in a 96-well plate in triplicate and incubated at room temperature for 5 minutes, after which the absorbance was read at a wavelength of 530 nm excitation and 586 nm emission using Tecan Infinite M200 PRO.
e effect of xylopic acid on CYP 1A1/1A2, 1A2, 2B1/2B2, and 3A4 was determined using fluorometric assays as described previously [19]. is was achieved by pipetting 70 μL of potassium phosphate buffer at a pH of 7.4 in triplicate into a 96-well plate followed by addition of 10 μL (2 μM) of each substrate (ethoxyresorufin (EROD), methoxyresorufin (MROD), pentoxyresorufin (PROD), and benzyloxyresorufin (BROD)). Subsequently, 10 μL of the microsomal fraction obtained from each treatment group was added to each well plate and preincubated at 37°C for 5 minutes, after which, 10 μL of nicotinamide adenine dinucleotide phosphate (NADPH) with a concentration of 100 μM was added to each of the wells and incubated at 37°C for 10, 20, and 30 minutes for CYPs 1A1/1A2, 1A2, 2B1/2B2, and 3A4, respectively. Plates were gently shaken after the addition of 40 μL of stopping solution (20% 0.5 M Tris and 80% acetonitrile) to each well. Fluorescence was determined at wavelengths of 586 nm emission and 530 nm excitation proceeding optimization of the protocol. e assay was repeated, and the mean values were used for computation.

In vitro Inhibitory Activity of XA on CYP 3A4.
Microsomes obtained from male rats pretreated with phenobarbitone were used in this assay. e standard inhibitor used was ketoconazole for CYP 3A4 and xylopic acid as the test compound. A cocktail was prepared for seven different concentrations for each standard inhibitor. e cocktail contained 3000 μl of 0.1 M potassium phosphate buffer (pH 7.4) which was pipetted into a 50 ml Eppendorf tube, followed by the addition of 600 μl of the substrate (benzyloxyresorufin) for CYP 3A4, and followed by the addition of 600 μl of the enzyme (microsomes). A volume of 80 μl was then pipetted from the cocktail into a 96-well plate in triplicate and then preincubated at 37°C for 5 minutes. Afterward, 10 μL of nicotinamide adenine dinucleotide phosphate (NADPH) with a concentration of (100 μM) was added to each of the wells followed by the addition of ketoconazole and xylopic acid to separate plates containing BROD. e plates were incubated at 37°C for 30 minutes for BROD. A volume of 40 μL of stopping solution (20% 0.5 M Tris and 80% acetonitrile) was finally added to each well. e plate was gently shaken, and its fluorescence read at wavelengths of 586 nm emission and 530 nm excitation [19]. e assay was repeated, and the mean values were used for computation.

Effect of XA on CYP 2D6.
e CYP 2D6-dextromethorphan-O-demethylation assay was used to determine the effect of xylopic acid on CYP 2D6. e assay was done by pipetting 350 μL of potassium phosphate buffer (pH 7.4) into Eppendorf tubes in triplicate. A volume of 50 μL of 0.25 mM dextromethorphan was added, followed by 50 μL of microsomes obtained from each treatment group. e mixture was preincubated at 37°C for 5 minutes in a water bath after which 50 μL of NADPH solution 100 μM was added. e mixture was further incubated for 45 minutes, followed by the addition of 100 μL of stopping solution (300 mM zinc sulphate heptahydrate). e mixture was centrifuged at 4,000 rpm for 15 min at room temperature, and the supernatant was collected in vials. Analysis of the supernatant was done using an isocratic HPLC (Shimadzu Prominence, Japan). e mobile phase consisted of 24% (v/v) acetonitrile and 0.1% (v/v) trimethylamine adjusted to pH 3.0 with perchloric acid. e carrier flow rate was 0.8 ml/min, and peaks were monitored at wavelengths of 280 nm excitation and 310 nm emission [19]. e assay was repeated, and the mean values were used for computation.

Effect of XA on CYP 2C9.
e CYP 2C9 diclofenac hydroxylation assay was used to determine the effect of xylopic acid on CYP 2C9 [26]. is assay measures the effect of XA on hydroxylation of diclofenac to 4-hydroxydiclofenac by CYP 2C9. is was done by pipetting 350 μL of potassium phosphate buffer (pH 7.4) into Eppendorf tubes followed by adding 50 μL of 0.05 mM diclofenac, after which 50 μL of the microsomal fraction obtained from each treatment group was added (in triplicate) and preincubated at 37°C for 5 minutes in a water bath. A volume of 50 μL of NADPH solution (100 μM) was added to each tube and further incubated in a water bath at 37°C for 10 minutes. e reaction was terminated by adding 200 μL of stopping solution (ice-cold methanol) to the mixture. e mixture was centrifuged at 12,000 rpm for 8 minutes at room temperature. e supernatants were collected in vials and analyzed using high-performance liquid chromatography [15]. e assay was repeated, and the mean values were used for computation.

Data Analysis.
For the in vivo testing, GraphPad Prism version 8 was used for all statistical analyses, and P values < 0.05 were considered statistically significant. All data obtained were expressed as a mean ± standard error of the mean. For the in vitro analysis, the column graphs were Advances in Pharmacological and Pharmaceutical Sciences subjected to one-way ANOVA with Tukey's post hoc test. Doses for 50% of the maximal effect (IC 50 ) for each drug were determined by using an iterative computer least squares method, with the following nonlinear regression (3parameter logistic) equation. e fitted midpoints (IC 50 s) of the curves were compared statistically using the F test.

Effect of Phenobarbitone and Xylopic Acid High and Low
Dose on CYP 1A2. All the treatment groups produced significant (P < 0.05) CYP 1A2 enzyme induction as shown in Figure 2. Administration of xylopic acid at a high dose of 100 mg/kg induced CYP 1A2 by 81.60% while low dose of 30 mg/kg by 77.20%. Phenobarbitone pretreatment induced CYP 1A2 by 70.10% as compared to the nontreatment group that served as the control.

Effect of Phenobarbitone and Xylopic Acid on CYP 1A1/1A2.
All the treatment groups had a significant (P < 0.05) effect on CYP 1A1/1A2, with phenobarbitone alone exhibiting the highest enzyme induction with a percentage activity of 139.9% (Figure 3). A high dose of xylopic acid (100 mg/kg) induced the enzyme resulting in an activity of 106.9% while xylopic acid low dose induced the enzyme with an activity of 118% compared to the negative control. e efficacy order for the various treatment groups on CYP 1A1/1A2 was phenobarbitone > xylopic acid low dose > xylopic acid high dose. Figure 4, the phenobarbitone pretreatment group had the most significant (P < 0.001) effect on CYP 2B1/2B2. XA low and high doses did not produce a significant effect on CYP 2B1/2B2 compared to the no treatment group. However, there was about a 2.5-fold increase in CYP 2B1/2B2 activity of phenobarbitone-treated animals compared to both doses of XA. Figure 5 illustrates the activity of phenobarbitone, ketoconazole, and XA low and high doses. Phenobarbitone induced CYP 3A4 with an about 6-fold increase in activity, while ketoconazole and XA low and high dose produced about 2-to 3-fold inhibition of CYP 3A4 activities. Comparatively, the inhibitory activity of XA high dose was approximately 1.36 times more than the standard ketoconazole. Figure 6 shows the effect of phenobarbitone and xylopic acid low and high dose on the biotransformation of diclofenac by CYP 2C9. All the treatment groups significantly induced the enzyme activity with percentage enzyme induction of 184.9%, 129.9%, and 151.8% for phenobarbitone, xylopic acid low dose, and xylopic acid high dose, respectively, as compared to the no-treatment group (P < 0.001).

Effect of Phenobarbitone and Xylopic Acid on CYP 2D6.
Effects of phenobarbitone, XA low dose, and XA high dose on CYP 2D6 are shown in Figure 7. e figure describes a significance in the difference between CYP 2D6 activity of the negative control experiment and the treated (P < 0.001) for phenobarbitone and XA high dose and a significance of (P < 0.05) in the case of XA low dose. Phenobarbitone, XA low dose, and XA high dose had activities of 48.6%, 24%, and 80.5%, respectively. XA high dose was 1.65 times more effective (P � 0.0029) than the standard phenobarbitone. and xylopic acid were 0.091 ± 0.002 μM and 1.30 ± 0.01 μM, respectively.

Effect of Xylopic
us, ketoconazole showed more potent inhibition of CYP 3A4 than xylopic acid.

Discussion
e study evaluated the ability of xylopic acid to influence the activity of hepatic CYPs in rats in vitro and in vivo. Interestingly, a recent study suggested that xylopic acid has a biphasic effect on rat hepatic CYP450 enzymes at low and high doses [24], but no specific enzymes were implicated in the biotransformation of the compound. Again, there are no studies on the effect of xylopic acid or the fruit of X. aethiopica (which contains xylopic acid) on human hepatic CYP450 enzymes although the fruits are heavily consumed as a spice in local dishes. erefore, there are no data on the metabolism of xylopic acid or its related kaurene diterpenes in rodents or humans, hence limiting these studies to implicate any possible metabolite of xylopic acid on the effects observed in these studies. Owing to the fact that X. aethiopica is heavily consumed, xylopic acid or its possible active metabolites can reach concentrations high enough to interact with other medications when taken concurrently. It was therefore imperative to identify the specific isoenzymes induced and inhibited on the administration of xylopic acid with a focus on CYP 1A1/1A2, 1A2, 2B1/2B2, 3A4, 2D6, and 2C9.
Xylopic acid induced CYP 1A2 in vivo at low and high doses, like the effect seen in phenobarbitone. Studies have shown that CYP 1A2 is responsible for the primary metabolism of theophylline and propranolol amongst others [28]. A study implicated cimetidine which is a histamine H 2 -receptor antagonist as an inhibitor of CYP 1A2 [28]. is suggested that the administration of xylopic acid with H 2 -receptor antagonists may result in drug-drug interaction.
CYP 2B1/2B2 is a phenobarbital-inducible member of the P450 xenobiotic-inducible superfamily involved in the hydroxylation of decanoic and other fatty acids [29]. e current study revealed that xylopic acid had no activity (induction/inhibition) on CYP 2B1/2B2. CYP 3A4 was inhibited by low and high doses of xylopic acid. e inhibition observed was weaker than that expressed by ketoconazole, a standard inhibitor of 3A4. CYP 3A4 accounts for 30-50% of drugs metabolized through type I enzymes [30]; therefore, it was paramount to investigate the effect of xylopic acid on CYP 3A4. Although the dose of xylopic acid used in the in vivo studies were 30 and 100 mg/kg, the amount absorbed into the bloodstream was not measured, but the concentration achieved in the blood can be high enough to produce food-drug interaction since this concentration was able to inhibit the CYP 3A4 enzymes.
Xylopic acid at low and high doses induced CYP 2C9. It is primarily expressed in the liver; a study suggests that its expression level is the second highest among CYP isoforms. It is responsible for the metabolic clearance of up to 15-20% of all drugs [31]. Some other substrates metabolized by this CYP aside from diclofenac include alkylating agents such as cyclophosphamide [32] and antiestrogenic drugs such as tamoxifen [33]. Treatment with rifampicin has been implicated in increased clearance of drugs metabolized by CYP 2C9 [32]. Drugs that inhibit this enzyme include fluconazole and sulfaphenazole. ere is evidence supporting the danger of drug-drug interaction when sulfaphenazole is added to a therapeutic regime that includes drugs with a low therapeutic index, such as warfarin [34]. is suggests that the use of xylopic acid with these inhibitors should be carefully considered.
Patients vary widely in their response to drugs. ey include individuals with no CYP 2D6 activity and individuals with genetically elevated CYP 2D6 activity, suggesting an altered response to drugs that are CYP 2D6 substrates. Drugs that inhibit CYP 2D6 activity are likely to increase the plasma concentrations of other medications, such as fluoxetine and paroxetine leading to drug toxicity [35].

Conclusion
Xylopic acid induced rat liver microsomal CYP 1A1/1A2, 1A2, 2D6, and 2C9; inhibited CYP 3A4; and did not affect CYP 2B1/2B2. Since these enzymes are implicated in the metabolism of several drugs, it is expected that XA can potentially influence the metabolism of other drugs through food-drug and drug-drug interactions.
Data Availability e data in support of the findings of this study may be requested and obtained from the corresponding author.

Conflicts of Interest
e authors declare they have no conflicts of interest.